Two very different bacterial pathogens, the Gram-negative Bacteroides fragilis and the Gram-positive Clostridium difficile, have been considered as textbook examples of strict anaerobes that inhabit the large intestine. B. fragilis is a normal inhabitant of the colon, but C. difficile is usually found in people whose normal microbiota has been compromised by antibiotic treatment. B. fragilis is an opportunistic pathogen mainly associated with escape from the intestine due to leakage into and formation of abscesses in the peritoneal cavity. C. difficile, on the other hand, is the primary cause of antibiotic-associated diarrhea and pseudomembranous colitis. Given that the normal habitat for these bacteria is the anaerobic environment of the large intestine, it is remarkable that our preliminary results have shown that mutants of B. fragilis and C. difficile arise that can grow in the presence of oxygen (up to 2%) and can survive exposure to even higher oxygen levels. Many of these mutants have oxe (oxygen-enabling) null mutations that lie in a gene called oxeA that is conserved in the two otherwise very dissimilar species. Moreover, many clinical isolates of B. fragilis derived from abscesses can grow in 1.5% oxygen and have oxeA mutations as well. This application seeks to explain these unexpected findings by addressing a fundamental biological question: Why do anaerobic pathogens normally express genes whose products prevent them from growing aerobically? Our working hypothesis is that the wild-type product(s) of the oxe genes promote growth or survival in the anaerobic colon whereas the oxe mutations enhance some aspects of pathogenesis in animal hosts. To test this hypothesis we will compare the colonization, virulence and transmission of B. fragilis and C. difficile wild-type and oxe mutant strains in animal models of disease. In addition, we will seek to uncover the biochemical mechanisms by which the oxe gene products prevent survival and growth in low levels of oxygen. The results are expected to reveal a novel and surprising aspect of anaerobic growth. PUBLIC HEALTH RELEVANCE: Bacteroides fragilis and Clostridium difficile are very different pathogenic bacteria that inhabit the anaerobic environment of the large intestine. This project seeks to reveal why both species express genes whose products prevent the bacteria from surviving and growing in even low concentrations of oxygen. The hypothesis to be tested is that successful colonization of the large intestine requires gene products whose activities decrease the ability of the cell to inactivate reactive oxygen species.